In recent years, mouse and human iPS cells have been established one after another. Yamanaka et al. identified genes expressed specifically in pluripotent cells such as ES cells and germ cells by analyzing the EST database, and conducted functional analyses using the knockout mouse technique and the like. Taking into consideration some reports by other research groups, they selected 24 genes as candidate substances that induce pluripotency (reprogram the nucleus) in somatic cells [WO 2007/069666 A1; Takahashi, K. and Yamanaka, S., Cell, 126: 663-676 (2006)]. They induced iPS cells by introducing these 24 genes into fibroblasts (MEF) from a reporter mouse wherein the neomycin resistance gene is knocked-in into the Fbx15 locus and forcing the cells to express these genes, by means of retrovirus. They proceeded to narrow the coverage of the genes that are most important to nuclear reprogramming by transferring 23 out of the 24 genes, and eventually identified the four genes Oct3/4, Sox2, Klf4 and c-Myc as essential factors for nuclear reprogramming in somatic cells [WO 2007/069666 A1; Takahashi, K. and Yamanaka, S., Cell, 126: 663-676 (2006)].
In addition, Yamanaka et al. succeeded in establishing iPS cells (Nanog iPS cells) that show almost the same gene expression and epigenetic modification profiles as those in embryonic stem (ES) cells and preparing a chimeric mouse, by producing a transgenic mouse wherein the green fluorescent protein (GFP) and puromycin-resistance genes are integrated into the locus of Nanog, whose expression is more localized in pluripotent cells than Fbx15 expression, forcing MEF derived from the mouse to express the above-mentioned four genes, and selecting puromycin-resistant and GFP-positive cells [Okita, K. et al., Nature, 448: 313-317 (2007)]. Thereafter, it was revealed that iPS cells could also be produced with three factors other than the c-Myc gene, which also contributes to the germline of chimeric mouse [Nakagawa, M. et al., Nat. Biotethnol., 26: 101-106 (2008)].
Furthermore, Yamanaka et al. succeeded in establishing iPS cells by introducing the same four genes or three genes as those used in the mouse into human skin fibroblasts [WO 2007/069666 A1; Takahashi, K. et al., Cell, 131: 861-872 (2007)]. Hence, it has been demonstrated that iPS cells comparable to ES cells in terms of pluripotency can be produced in both humans and mice, by introducing defined factors into somatic cells.
Of the four genes Oct3/4, Sox2, Klf4 and c-Myc, Oct3/4 and Sox2 are reportedly essential for maintaining self-renewal and pluripotency of ES cells, and c-Myc has also been reported to be involved in maintaining self-renewal and pluripotency of ES cells. Meanwhile, Klf4 belongs to the family of Krüppel-like factor (Klf), a transcriptional factor that controls various biological processes, including proliferation, differentiation, development, and apoptosis [McConnell, B. B. et al., Bioassays, 29: 549-557 (2007)], but details of its functions remain unclear. Epiblast stem cells (EpiSC) established from the epiblast of post-implantation embryo, unlike ES cells, are incapable of forming a chimeric embryo even when injected into a host blastocyst. In EpiSC, however, Oct3/4 and Sox2 are expressed at levels similar to those in ES cells, whereas the Klf4 gene is expressed at remarkably lower levels. Recently, it was reported that by transferring the Klf4 gene alone into EpiSC, a nature similar to that of ES cells can be acquired [Guo, G. et al., Development, 136: 1063-1069 (2009)].
Since ES cells exhibit no morphological changes even when Klf4 is knocked down by RNAi [Nakatake, Y. et al., Mol. Cell. Biol., 26: 7772-7782 (2006)], however, Klf4 may be unessential to the maintenance of the undifferentiated state of ES cells. Yamanaka et al. hypothesized that the same four genes could be substituted by other genes belonging to the same respective families, and showed that iPS cells could be established even when Klf4 was replaced with Klf1, Klf2 or Klf5 [WO 2007/069666 A1; Nakagawa, M. et al., Nat. Biotethnol., 26: 101-106 (2008)]. A group of Thomson et al. reported that human iPS cells could be generated using Nanog and Lin28 in place of Klf4 and c-Myc [WO 2008/118820 A2; Yu, J. et al., Science, 318: 1917-1920 (2007)]; the function of Klf4 can be thought to have many common aspects compared with Nanog.
When ES cells are treated with retinoic acid to induce their differentiation, not only expression of Klf4, but also expression of Klf2 and Klf5 decrease. Taking note of this fact, Jiang et al. knocked down Klf2, Klf4 and Klf5 simultaneously, and found that differentiation was induced in the ES cells, showing that at least some of the members of the Klf family, such as Klf2 and Klf5, can functionally substitute for Klf4 in ES cells [Jiang, J. et al., Nat. Cell Biol., 10: 353-360 (2008)]. They proceeded to transfer the Klf2 or Klf5 gene, or other transcriptional factors and epigenetic regulatory factor, along with the three genes Oct3/4, Sox2 and c-Myc, into MEF; they confirmed that Klf2 and Klf5 can substitute for Klf4, and found that Esrrb, an orphan nuclear receptor similar to estrogen receptors, is also capable of substituting for Klf4 [Feng, B. et al., Nat. Cell Biol., 11: 197-203 (2009)].